Method and apparatus for measuring a state variable

ABSTRACT

A method is provided for measuring a state variable of a biological cell ( 3 ) located in a nutrient medium ( 2 ) and supported on and adhering to a support area ( 5 ). Within the support area ( 5 ) for the cell ( 3 ) and at a distance from the support area edge, an opening is made in the membrane of the cell ( 3 ). The edge of the cell membrane that surrounds the opening and adheres to the support area ( 5 ) seals off the liquid found inside the cell ( 3 ) from the nutrient medium ( 2 ). Through the opening the state variable ( 2 ) is measured. An apparatus for performing the method is also provided.

BACKGROUND OF THE INVENTION

The invention involves a method for measuring at least one statevariable of a biological cell located in a nutrient medium, the cellbeing supported on and adhering to a support area, wherein at least oneopening is made in the membrane of the cell for measuring the statevariable. The invention further involves an apparatus for measuring atleast one state variable of at least one biological cell located in anutrient medium, the apparatus including an object carrier (specimenslide) having a support area, on which the cell can be supported in anadherent manner, and at least one measuring probe that can be broughtinto contact with the cell liquid located inside the cell for measuringthe state variable, wherein the measuring probe is connected or combinedwith a measurement amplifier.

From the book, Human Physiology, Schmidt; Thews (publisher), 23^(rd)edition (1987), pages 20-21, an apparatus of this type is already known,which has a suction device connected to a hollow needle with an innercavity that has an opening on the open end of the hollow needle. In theinner cavity of the hollow needle, a measuring probe is arranged formeasuring the cell potential of the cell. With this device, operatingaccording to the so-called patch-clamp process, the hollow needle isapplied on the outside of the cell membrane in order to bring themeasuring electrode into contact with the cell liquid by the openinglocated on the free end of the hollow needle, in order to then generatea partial vacuum in the inner cavity of the hollow needle using thesuction device. With this partial vacuum a piece of the cell membrane,located in front of the opening of the hollow needle, is torn out of themembrane structure. Via the resulting opening in the cell membrane, theions located in the cell liquid get into an electrolyte located in thehollow needle and from there to the measuring probe. A referenceelectrode functions for determining a reference potential.

The previously known method and associated apparatus have thedisadvantage that, for positioning of the hollow needle on the cell, amicromanipulator is necessary. This results in a comparativelycomplicated and expensive device. Moreover, the accessibility of thecells located on the specimen slide is greatly restricted by themicromanipulator. The process and apparatus are thus suitable only foran investigation of individual, or at most for a simultaneousinvestigation of a small number of, cells located on the specimen slide.

From U.S. Pat. No. 4,461,304 an apparatus is further known, which has aneedle-shaped tip for making an opening in a cell membrane. On the tip aplurality of sensors is arranged for neurophysiological investigations.Even with this device, for positioning of the tip on the cell, amicromanipulator is necessary.

From published European and German patent applications EP 0 689 051 A2;DE 197 12 309 A1; and EP 0 585 933 A2 and German patent DE 195 29 371C2, apparatus are already known for measuring a cell potential, whichhave a specimen slide having a plurality of microelectrodes arranged inmatrix form, which can be brought into connection with the outside ofthe membrane of a cell to be investigated. These devices make possible,however, only an extracellular measurement of the cell potential, sinceno opening is made in the cell membrane.

From published German patent applications DE 195 36 389 A1 and DE 195 36384 A1 methods are already known for measuring a state variable, inwhich a biological component is contacted. Also with this method, anopening is not made in the biological component.

In German published patent application DE 38 16 458 A1 a microelectrodeis further described, which can be used for a potentiometric oramperometric measurement in the biochemical and medical fields.

German patent DE 44 22 049 C2 discloses an ultra-microelectrode arrayfor chemical and biochemical analyses, which has several pyramids orcone-shaped electrode tips on a substrate. According to statements ofthe patent document, the ultra-microelectrode array can be inserted intoelectrode structures for the measurement of oxygen according to Clark.

From U.S. Pat. No. 5,173,158, moreover, a process outside of the genericconcept is known for generating new cells, in which cells of a firsttype located in a liquid are supported by a partial vacuum orhydrostatic pressure on a support area of a porous layer, such that thecells engage with a component in the pores of the porous layer. Theporous layer with the cells is arranged between electrode platesbordering on the liquid to which an electric voltage is applied, whichopens the cell membrane of the cells in the component that engages withthe pores. Thereafter, cells of a second type are brought into theinsides of the cells of the first type through these openings.

SUMMARY OF THE INVENTION

Thus, an object of the present invention is to create a method andapparatus of the type mentioned at the outset, which makes possible asimple measurement of a state variable of the cells. In particular, acostly manual positioning of a hollow needle on the cells to beinvestigated should be avoided.

This object is achieved with respect to the method in that the openingin the cell membrane is made within the support area of the cell andspaced from the support edge, and through this opening the statevariable is measured.

In this way it is possible to arrange a poration agent or a porationtool, which is used for making the cell membrane opening, in the supportarea of the cells on the specimen slide so that the cell is alsopositioned simultaneously on the poration agent or poration tool when itis supported on the support area. In this way, a costly manualpositioning of a poration tool can be omitted. Since the opening isformed in the cell membrane within the support area of the cells and ata distance from the edge of the support area, the membrane area of thecells, which surrounds the opening and is adheringly attached to thesupport area, seals off the opening from the nutrient liquid. The cellliquid located inside the cell is thus electrically insulated to thegreatest extent possible from nutrient liquid. The measured statevariable of the cells can, for example, be an ion concentration, a gasquantity, a temperature or any other desired physical, chemical orbiological characteristic of a cell.

In order to measure the cell potential of the cells, the electricalvoltage between the cell liquid and the nutrient medium can be measuredthrough the opening formed in the cell membrane. Using this process, forexample, electrical signals transmitted between nerve cells can betested. Using this process, electric d.c. and/or a.c. voltagepotentials, especially potentials that change quickly in time, can bemeasured.

In an especially advantageous embodiment of the invention, the openingcan be made using electroporation of the cell membrane. For theperformance of the process, for example, an electroporation-electrodecan be arranged in the support area for the cell, on which the cell issupported in an adherent manner. In order to form the opening in thecell membrane, an electric voltage then only needs to be applied betweenthe electroporation electrode and the nutrient medium, which causes anelectrical current flow that opens the cell membrane. After switchingoff the electroporation voltage, the electric potential can be measuredthrough the opening of the cell membrane between the cell liquid and thenutrient medium. For this purpose, optionally, the electrode used forelectroporation can also be used to measure the cell potential, so thatthe electrode performs a double function.

In another embodiment of the invention, at least one mechanical impulseis applied in order to form the opening in the cell membrane on aportion of the cell membrane. In this process, this portion of the cellmembrane detaches from the membrane structure. Optionally , an impulsesequence can also be applied with several individual impulses.

It is especially advantageous when the opening is made in the cellmembrane using ultrasound. In this process, it is even possible that theultrasound is focused on the area of the cell membrane to be openedand/or that several ultrasonic waves are superimposed so that theiroscillations are superimposed in the area of the cell membrane to beopened, to produce an oscillation having an increased amplitude. Thecell membrane can be opened in this way without contact.

A contact-free opening of the cell membrane can, however, also occur ina manner such that a portion of the cell membrane is irradiated with ahigh-energy radiation, especially with laser radiation. In this process,the wavelength of the radiation is preferably selected such that thecell membrane absorbs the radiation well. Functionally, the radiation iscoupled into the cell in the support area of the cell. Optionally, alaser beam can, however, also be coupled into the cell outside thesupport area, where in a membrane area located there a small couplingopening is first made, through which the laser beam is then projectedthrough the inside of the cell onto a membrane area located in thesupport area of the cell, in order to cut out a portion of the membranefrom the membrane structure by sweeping of the laser beam around thecoupling opening.

In another embodiment of the process, the opening is made by the actionof a chemical substance in the cell membrane. As a poration agent, forexample, a Perforin or Triton® can be used.

It is especially advantageous if an electric and/or chemical substanceand/or a substance that can be activated by radiation is used and ifthis substance is activated in order to make the opening in the cellmembrane by the action of radiation and/or an electric field. Thesubstance is thus activated by the supply of energy. In this process,for example, free radicals are generated, which destroy the portion ofthe cell membrane that is to be opened. In the inactive condition, thesubstance acts, for the most part, neutrally relative the cell, so thatit has practically no influence on the support of the cell on thesupport area. A chemical substance can also be used which is chemicallyactivated by the addition of another substance.

Another embodiment of the process provides that a portion of the cellmembrane to be opened is disengaged from the membrane structure byimpingement with a partial pressure and/or an excess pressure. In thisprocess, for example, in a specimen slide that has the support area, asmall opening can be provided within the support area, through which thecell is suctioned to such an extent that the membrane area located infront of the opening is torn out of the membrane structure. In order toopen the cell membrane, action can be made through the opening, butalso, however, an excess pressure pulse can be exerted on a membranearea of the cell.

It is advantageous that the cell be fixed on the support area by asuction force. In this way, the adhering of the cell on the support areaof a specimen slide is improved. The suction force is measured selectedsuch that the cell membrane is not mechanically damaged by the suctionforce.

It is advantageous when, after making the opening in the cell membrane,cell liquid is removed from the cell through the opening and examined.In this way, it is possible to detect additional cell quantities thatare only measured with difficulty inside the cell. Thus, additionalinformation about the physiology of the cell can be obtained.

It is especially advantageous when, after making the opening in the cellmembrane, an intracellular manipulation can be performed through theopening, in particular, a medicine and/or a foreign substance and/or abiological substance is brought into the interior of the cell. Theeffects of the intracellular manipulation can then be observed bymeasuring a state variable of the cell. Optionally, the intracellularmanipulation can also be performed, however, without a state variable ofthe cell being measured through the opening.

In regards to the apparatus, the previously mentioned purpose isachieved in that the measuring probe and an electric insulatorsurrounding it are arranged within the support area, such that the cellcan be supported on the insulator to seal it from the nutrient medium,and that to open the membrane of the cell, at least one poration tool isarranged in the support area in the area of the measuring probe.

Since the poration tool is arranged within the support area, the cellcan automatically be supported on the poration tool located in thesupport area. In an advantageous way, a costly manual positioning of theporation tool on the cell can thus be omitted. Also, auxiliary devices,such as micromanipulators, are not necessary. In this way it is possibleto measure cell quantities at the same time on several cells that arearranged in close proximity to one another. After making the opening inthe cell membrane, the edge of the cell membrane surrounding the openingstays in contact with the electric insulator and seals off the openingfrom the nutrient medium. In this way, the cell liquid located insidethe cell is electrically well-insulated from the nutrient medium, sothat the cell quantity can be measured through the opening of the cellmembrane.

It is advantageous when the poration tool is arranged substantiallyconcentrically around the measuring probe. The measuring probe can thencome into good contact with the cell liquid located inside of the afterthe opening is made in the cell membrane.

In an embodiment which is provided for measuring the potential of thecell, the measuring probe is a measuring electrode, to which isallocated at least one reference electrode that can be brought intocontact with the nutrient medium. With a device of this type, thedifference in potential between the cell liquid and the nutrient mediumcan be measured in a simple way.

It is especially advantageous when the measurement electrode also is anelectroporation electrode which can be connected to an electric voltagesource for the electroporation of cell membrane, and when theelectroporation electrode has at least one active electrode area that isarranged within the support area and which is surrounded by the electricinsulator. The measuring electrode is thus simultaneously also theporation tool, and fulfills a double function, such that a deviceresults that is constructed in an especially simply manner. Theelectrode connected to the measurement amplifier can, for example, beapplied briefly at the potential of the voltage source using anelectronic switch. Optionally, a change-over switch can also beprovided, by which the measuring electrode can be selectively connectedone after the other to the measurement amplifier or the voltage source.The measuring and electroporation electrode is arranged within thesupport area of the specimen slide so that a cell supported so as toadhere to the insulator can also optionally be supported on the activeelectrode area of the electrode or at least approach it until reachingthe active area of an electric field that extends from the electrode.When the electroporation voltage is applied to the electrode, anelectric current flows which makes an opening in the cell membrane. Inthe process, the edge of the cell membrane surrounding the opening staysin contact with the electric insulator and seals off the opening fromthe nutrient medium. The insulator resistance is independent of the celltype and is preferably larger than 10 megaohms. An offset of thepotential between the nutrient medium and the cell liquid is thereby forthe most part stopped. After the creation of the opening in the cellmembrane, the measuring electrode is separated from the electroporationvoltage source, so that then the cell potential is applied on themeasuring electrode that is in contact with the cell liquid and can bemeasured with the measurement amplifier. The device has a simpleconstruction and makes possible, for the most part, an automated cellpotential measurement.

In an advantageous embodiment of the invention it is provided that theporation tool is an electroporation electrode that is set off at adistance from the measuring probe. The electrode can be connected to anelectric voltage source for the electroporation of the cell membrane.The electroporation electrode has at least one active electrode areaarranged within the support area and surrounded by the electricinsulator. The electroporation electrode is thus separated from themeasuring probe. In this way, the effect of the capacitance of thesupply lines from the voltage source to the electroporation electrode isreduced to the measurement signal determined by the measuring probe.Thus, changes of the cell potential that are rapid in time, as occurs innerve cells for example, can be measured even better.

In an especially advantageous further embodiment of the invention, it isprovided that the active electrode area of the electroporation electrodehas at least one sharp tip or edge and is preferably arranged to projectbeyond the surface plane of the support area. The active electrode areacan thus contact the cell membrane of a cell supported on the supportarea on the outside. In this way, a good electrical contact between theelectrode and the cell membrane is possible. The projecting activeelectrode area additionally allows a good electrical contact with thecell liquid located inside the cell through the opening of the cellmembrane and, above and beyond that, acts to oppose a closing, throughcell repair mechanisms, of the opening made by electroporosis in thecell membrane.

It is advantageous if the electroporation electrode is constructed as ahollow electrode which has an inner cavity with an opening located onthe surface of the support area, and if the measuring electrode is a rodelectrode arranged in the inner cavity of the electroporation electrode,the free end of the rod electrode preferably extending up to the openingof the inner cavity. Inside the hollow electrode, an electrolyte can bearranged which can correspond, for example, to the nutrient medium, sothat after the opening of the cell wall, charged particles, especiallyions, contained in the cell liquid can migrate through this electrolyteto the electrode. Thus, a larger electrode surface can come into contactwith the charged particles. The electric contact between the measuringelectrode and the cell liquid is improved by this.

It is especially advantageous when immediately adjacent to theelectroporation-electrode a switching component, in particular a staticswitch, is arranged with which the electroporation electrode can beconnected to the electroporation voltage source. The connection linebetween the electroporation electrode and the electroporation voltagesource can then be uninterrupted closely adjacent to the electrode, sothat the parasitic capacitance of this connection line is uncoupled fromthe measuring probe during the measurement of the cell potential. Inthis way, cell potential voltages that change quickly in time can bemeasured more exactly. Preferably, the static switch is a low-noisejunction-field effect-transistor.

In an advantageous embodiment of the apparatus, the poration tool foropening the cell membrane can be moved using at least one actuator, inparticular a piezo element, across the surface of the support arearelative to the specimen slide. With this device, the opening is thusmade mechanically in the cell membrane. In the process, the porationtool can even be alternately moved away from and toward the cellmembrane. For this purpose, the actuator can be connected with a drivedevice for generating an ultrasonic oscillation. The poration tool canbe alternately moved relative to the specimen slide in a direction thatruns perpendicular or in a direction that is oblique to the surface ofthe support area.

It is advantageous when the poration tool has at least one sharp tip oredge that can be brought into contact with the membrane of the cell. Thecell membrane can then be better opened by moving the poration tool.

It is especially advantageous when the measuring probe is at the sametime also the poration tool and for this can be moved, using theactuator or piezo element, across the surface of the support arearelative to the specimen slide. The measuring probe then fulfills adouble function, such that an additional poration tool can be omitted.

In another advantageous embodiment, the specimen slide has an opticalwindow in the area of the measuring probe, which is arranged in the beampath of a laser beam in order to open the cell membrane. Using thisdevice, a portion of the cell membrane can be irradiated briefly withhigh-energy optical radiation, where this area is heated to such anextent, that an opening is made in the cell membrane.

It is especially advantageous when, in order to generate the laser beam,a laser diode is integrated into the specimen slide. In this process,the laser diode can even be arranged directly behind the opening, sothat the laser beam can be coupled directly and thus, for the most part,free of losses into the cell membrane supported on the support area.

Functionally, the measuring probe is arranged substantiallyconcentrically around the optical window. The measuring probe can thencome into better contact with the cell liquid after opening the cellmembrane.

In an advantageous embodiment is provided that the poration tool has, inorder to open the cell membrane, a chemical substance and/or at leastone outlet opening connected to the supply channel for a chemicalsubstance. The opening can thus also be made in the cell membranechemically, where as a poration agent, for example, Perforin or Triton®can be used.

In another embodiment the poration tool has at least one channel thatopens into the support area, by the use of which a portion of the cellmembrane can be impinged in order to make the opening in the cellmembrane with a partial vacuum and/or excess pressure. In a device ofthis type, the opening is thus made in the cell membrane using a partialvacuum or excess pressure, where the partial vacuum or excess pressureis switched off after the opening of the cell membrane. During anopening of the cell membrane by partial vacuum, a suction of the cellliquid out of the inside of the cell is prevented to the greatest extentpossible. Correspondingly, during opening of the cell membrane usingexcess pressure, it is prevented that a medium located in the channel,that is preferably a fluid, can enter into the inside of the cell. Inorder to switch off the partial vacuum or excess pressure, for example apressure change occurring during the opening of the cell membrane in thechannel can be determined. The partial vacuum or excess pressure can begenerated using a suitable auxiliary device, for example a pump, orhydrostatically. In an advantageous way, the channel can also be usedprior to supporting the cell, in order to suction off nutrient mediumfrom the support area, so that a current occurs in the nutrient medium,which conducts the cells located therein to the opening of the channelarranged in the area of the measurement electrode.

It is especially advantageous when the measuring probe is constructed asa hollow sensor that is installed into the surface of the specimenslide, and has at least one inner cavity, wherein the inner cavity hasan opening on the surface of the support area. In the inside of thehollow electrode, an electrolyte can be arranged, so that chargedparticles contained in the cell liquid can migrate through theelectrolyte to the measuring probe. Thus, a larger surface of themeasuring probe can come contact with the charged particles, whichimproves the electrical contact between the measuring electrode and thecell liquid.

An advantageous embodiment provides that the electric insulator withinthe support area has a projection that projects beyond the surface planeof the support area, and that the measuring probe is arranged on thefree end of the projection, which faces away from the surface of thesupport area. In this way, a good electrical and/or mechanical contactresults between the measuring probe and the cell membrane.

Functionally, it is provided that the cross-section of the projectionproceeding out from the surface plane of the support area tapers to thepoint that projects furthest out. The cell then adheres better with itsmembrane to the projection of the electric insulator. In addition, theinsulator can, in production engineering in the manufacture of thespecimen slide, be better applied as a coating on the area of theprojection that tapers.

In an advantageous further embodiment of the invention, it is providedthat the specimen slide has a profile in the support area, which has atleast one profile recess and/or a profile projection that surrounds themeasuring probe. In this way, a better seal of the cell liquid isachieved against the nutrient medium by the cell membrane that adheresto the insulator.

It is advantageous when the profile recess and/or profile projection isinterrupted in the extension direction by at least one gap. The cell canthen better adhere to the surface of the specimen slide in the area ofthe profile. The profile projection or profile recess can, for example,have a web structure or a structure in the manner of a checkerboardpattern.

It is especially advantageous if the profile recess and/or the profileprojection is constructed in a ring-shape, and if preferably severalsuch ring-shaped profile recesses and/or profile projections arearranged essentially concentrically to the measuring probe. Thus,radially to the measuring probe, several profile recesses and/orprojections are connected behind each other or interlaced within eachother, so that the cell liquid is even better sealed off from thenutrient medium.

A preferred embodiment of the invention provides that the insulator isan insulation layer arranged on the surface of the profile. In anadvantageous manner, the path for a leak current flowing on the surfaceof the insulator from the cell liquid to the nutrient medium is enlargedby the insulation layer profiled in this way, so that the measuringprobe or the measuring electrode is insulated even better from thenutrient medium.

Another embodiment provides that the profile projection(s) is (are)mounted on the surface of the insulator. The specimen slide can then bemanufactured in a more simple way by production engineering.

It is especially advantageous when in the support area of the specimenslide on its surface, at least one coating that has a cell adhesionprotein and/or a hydrophilic coating is arranged. The cell membrane thenadheres better to the specimen slide. The cell adhesion coating can forexample have laminin, fibronectin, or poly-L-lysine. Possibly, ahydrophobic coating can also be arranged on the edge of the support areathat borders the electrode, with bonding positions for the hydrophobiclipids located in the cell membrane.

It is advantageous when as a mechanical guide for the cells, boundarywalls are arranged on both sides of the measuring probe, whichpreferably delimit a groove-like guide channel. In this manner, themeasuring electrode(s) is (are) preferably arranged in the middlebetween the boundary walls at the base of the groove of the guidechannel, so that cells located in the guide channel can move essentiallyonly in the extension direction of the guide channel and then inevitablycome into contact with the measuring probe.

In an especially advantageous further embodiment of the invention, afield-effect transistor (FET), in particular a junction-field-effecttransistor (J-FET) is arranged adjacent to the measuring probe, and themeasuring probe is connected for the impedance transformation of themeasuring signal to the gate of the FET. In the process, the coupling ofthe measuring electrode onto the gate is done capacitively in a metaloxide-field effect transistor (MOS-FET), wherein the measuring electrodeis preferably arranged directly above the gate of the MOS-FET that isinstalled into the surface of the specimen slide. In an advantageousway, a junction-FET makes possible a high-ohmic, but nevertheless,low-noise coupling of an intracellular electrical signal. The low inputcapacitance of the junction-FET allows, in particular, even for rapidchanges in the cell potential, obtaining a measuring signal that is, forthe most part, free from feedback. By the impedance conversion directlyat the measuring site, the screening cost for the connection lines fromthe measuring electrode to a measurement amplifier and/or an evaluationdevice can be reduced. The field effect transistors manufactured by themanufacturing processes known in semiconductor technology allow, beyondthat, a high integration density.

It is advantageous when at least one liquid channel opens in the supportarea of the specimen slide, adjacent to the measuring probe, preferablyin an area surrounded by this. Through this liquid channel, after theopening of the cell membrane, a small quantity of cell liquid can besuctioned off and/or a biological substance, for example a gene, and/ora foreign substance, a medication or the like, whose effect on the cellis to be investigated, can be added to the cell liquid. To add asubstance, the device can optionally also be used without a measuringprobe.

Expediently, in the course of the liquid channel, a micropump preferablyintegrated in the specimen slide is arranged. Optionally, severalmicropumps can even be allocated to one liquid channel which, forexample, can respectively be connected to a cavity located in thespecimen slide to support a liquid or a medium that is to becytologically tested.

It is especially advantageous when within the liquid channel, preferablyin a wall of the liquid channel, at least one microsensor is arrangedfor measuring a quantity of the cell. In this way, additionalintracellular parameters, such as ion concentrations, gas contents,enzyme and/or protein concentrations, can be determined.

It is advantageous when, in order to generate an electric field thatconducts the cell to the measuring probe, at least one additionalelectrode is arranged in the support area and/or adjacent to it. Anelectric field can thereby be generated on the surface of the specimenslide, which exerts a force on the biological cells, whose dielectricconstants are distinguished from those of the nutrient medium in whichthey are arranged. This force guides the cells to the measuring probe.

An especially advantageous further embodiment of the invention providesthat in the support area several measuring probes and electroporationelectrodes are preferably arranged as an array, and that to each ofthese measuring probes at least one poration tool is allocated. Such adevice allows a locationally triggered measurement of the cell potentialon a cell population. In this process, the cell potential can bemeasured on many cells that are close to each other at the same time. Inthis way, it is possible, for example, to perform quasi static cellmembrane potential measurements on nerve or tumor cells, in order tomonitor their electric activity. In this way, it is even possible tomonitor the information transfer between the cells, through thelocationally triggered and time-triggered measurement of the cellpotential, in a cell composite with nerve cells connected throughsynapses. Optionally, a multiplexer can also be integrated into thespecimen slide, by which several measurement and/or electroporationelectrodes can be connected alternatingly one after the other with ameasurement signal guide, such that the number of the supply lines tothe specimen slide constructed as a sensor chip is correspondinglyreduced.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings embodiment(s) which are presentlypreferred. It should be understood, however, that the invention is notlimited to the precise arrangements and instrumentalities shown. In thedrawings, which are schematically drawn:

FIG. 1 is a longitudinal section through a measuring and electroporationelectrode arranged in the support area of a specimen slide andselectively connectable with a voltage source and a measuring amplifierusing a change-over switch;

FIG. 2 is a plan view of the measuring electrode according to FIG. 1;

FIG. 3 is a representation similar to FIG. 1, where, however, separateelectrodes are provided for the measurement and the electroporation;

FIG. 4 is a plan view of the measuring and electroporation electrodesaccording to FIG. 3;

FIG. 5 is a representation similar to FIG. 1, where, however, theelectrode is constructed as a hollow electrode in the shape of atruncated cone with cylindrical inner cavity;

FIG. 6 is a plan view of the electrode according to FIG. 5;

FIG. 7 is a representation similar to FIG. 5, where, however, theelectrode has an essentially cylindrical shape;

FIG. 8 is a plan view of the electrode according to FIG. 7;

FIG. 9 is a longitudinal section of a device in which the measuring andelectroporation electrode is electrically coupled to the gate of aJ-FET;

FIG. 10 is a representation similar to FIG. 9, where, however, adjacentto the electrode, an additional field effect transistor is arrangedfunctioning as a switch, by which the electrode can be connected to anelectroporation voltage source;

FIG. 11 is a longitudinal section of an apparatus with a specimen slidehaving an electrode on which is adherently supported a biological celllocated in a nutrient medium;

FIG. 12 is a plan view of the specimen slide shown in FIG. 11;

FIG. 13 is a longitudinal section through a specimen slide, which has asurface profile surrounding the electrode, on which an electricinsulation layer is mounted;

FIG. 14 is a plan view of the specimen slide according to FIG. 13;

FIGS. 15 and 16 are views similar to FIG. 14, where, however, thespecimen slide has another surface profile;

FIG. 17 is a plan view of a specimen slide that has an array withseveral measuring and electroporation electrodes;

FIG. 18 is a longitudinal section through a specimen slide, which has anelectrode arranged between two boundary walls within a surface profile;

FIG. 19 is a longitudinal section through a measuring andelectroporation electrode that can be moved using a piezo element and isarranged in the support area of a specimen slide;

FIG. 20 is a longitudinal section through an electroporation electrodethat has an optical window and is arranged in the support area of aspecimen slide, behind which a laser diode is arranged;

FIG. 21 is a representation similar to FIG. 20, where, however, anexternal laser is used, which has a laser beam that is coupled into theoptical window; and

FIG. 22 is a representation similar to FIG. 5, where, however, throughthe electrode a liquid channel leads to the support area and whereseveral measuring probes are arranged in the course of the liquidchannel.

DETAILED DESCRIPTION OF THE INVENTION

An apparatus, indicated as a whole by 1 for measuring the cell potentialof a biological cell 3 (shown in FIG. 11, but for sake of clarity, notshown in the other figures) located in a nutrient medium 2, has aspecimen slide 4 that has a support area 5, on which the cell 3 can besupported in an adherent manner. The cell 3 is thus immobilized on thespecimen slide 4 and adheres to the support area 5. In the embodimentsaccording to FIGS. 1 and 2 as well as 5 to 17, the specimen slide 4 haswithin the support area 5 a measuring and electroporation electrode 6,7,which has an active electrode area 8 that projects beyond the surfaceplane of the support area 5. In the support area 5, an electricinsulator 9 is arranged surrounding the active electrode area 8, onwhich insulator the cell 3 can be mounted to seal off the nutrientmedium 2.

In the embodiments according to FIGS. 1, 5, 7 and 11, the electrode canbe connected via a conductor path 10 integrated in the specimen slide toa change-over switch 11 with which it can be selectively connected to ameasurement amplifier 12 and an electroporation voltage source 43. Inorder to measure the cell potential, the electrode 6,7 is first broughtinto contact with the cell liquid located inside the cell 3. To do this,an electric voltage is applied between the electrode 6,7 and thenutrient medium 2, wherein the electrode 6,7 is connected via thechange-over switch 11 to the electroporation voltage source 43. Anelectric current thus flows over the electrode 6,7 into the cellmembrane, whereby in the area of the electrode 6,7 an opening is made inthe cell membrane, and the active electrode area 8 penetrates throughthis opening into the cell 3. The electrode 6,7 thereby comes intocontact with the cell liquid.

After making the opening in the cell membrane, the electrode 6,7 isconnected with the input of the measurement amplifier 12. The outlet ofthe measurement amplifier 12 is connected with a connection contact 13.An additional connection contact 14 is connected to a referenceelectrode 15, which is in electrically conducting contact with thenutrient medium 2. Between the connection contacts 13 and 14 an electricvoltage arises, which is a measure of the cell potential of the cell 3.Onto the connection contacts 13 and 14 can be connected, for example, adisplay and evaluation device . The opening made in the cell 3 using theelectroporation electrode 6,7 is sealed off from the nutrient medium 2by the cell membrane area surrounding the opening, where it adheres onthe specimen slide 4. A potential equalization between the potential ofthe electrode 6,7 and the nutrient medium 2 is thereby prevented.

In the embodiments according to FIGS. 1, 9 to 11 and 13, the electrode6,7 is constructed in an approximately cone shape, wherein the activeelectrode area 8 projecting beyond the surface plane of the support areais arranged on the tip of the cone and is constructed as a sharp tip.During the application of an electroporation voltage on the electrode6,7, a particularly high electric field strength thereby arises in theactive electrode area 8.

In the embodiments according to FIGS. 5 and 8, the electrode is a hollowelectrode, which is installed with its active electrode area 8projecting beyond the surface of the support area 5 in the surface ofthe specimen slide 4. The electrode 6,7 according to FIG. 5 isessentially in the shape of a truncated cone and the electrode 6,7according to FIG. 7 is essentially constructed as a cylindrical sheath,wherein the axis of symmetry of the electrode 6,7 is arrangedrespectively approximately perpendicular to the surface plane of thespecimen slide 4 in the support area 5. The hollow electrode has aninner cavity 16 filled with the nutrient medium 2, which has an openingon the surface of the support area 5. Through this opening, when theelectrode 6,7 is arranged in the cell membrane opening, electricallycharged particles from the cell liquid can reach into the inner cavity16 and come into contact with the inner wall of the electrode 6,7bordering the inner cavity 16. In contrast to the embodiment of FIG. 1,a larger active measuring electrode surface results, such that theelectrical contact resistance between the electrode 6,7 and the cellliquid is reduced.

As can be recognized especially well from FIGS. 5 and 7, the open end ofthe electrode 6,7 bordering the opening has a sharp ring edge, the crosssection of which is constructed as a tip and tapers away from thesurface plane of the specimen slide to the point on the electrode 6,7projecting furthest away. During the application of an electric voltageon the electroporation electrode 6,7 in the active electrode area 8, acomparatively high field strength thereby results, which makes theopening of the cell membrane easier.

In the embodiment according to FIGS. 3 and 4, a measuring electrode 6and an electroporation electrode 7 separated from it are arranged in thesupport area 5 of the specimen slide 4. The electroporation electrode 7is connected to an electroporation voltage source 43 via a conductorpath 17 integrated in the specimen slide 4, for making an opening in thecell membrane of a cell that adheres to the support area 5. Anadditional conductor path 18 connects the measuring electrode 6 to theinput of a measurement amplifier 12. Otherwise, the structurecorresponds for the most part to the specimen slide according to FIG. 5.The electroporation electrode 7 has approximately the shape of atruncated cone, and has a cylindrical inner cavity, the cylinder axis ofwhich approximately corresponds with the axis of symmetry of thetruncated cone. On the free end of the electroporation electrode 7, theinner cavity has an opening that leads to the surface of the specimenslide 4. The measuring electrode 6 is constructed as a rod electrodewhich is arranged approximately concentrically in the inner cavity ofthe electroporation electrode 7 and with its free end extends up to theopening of the inner cavity. In order to uncouple the measuringelectrode 6 from the parasitic capacitiance of the electroporationelectrode 7 and its supply line, and to increase the electric resistancebetween the measuring electrode 6 and the electroporation electrode 7,an insulator layer 19 can be arranged on the inner wall of theelectroporation electrode 7 which delimits the inner cavity 16. Theinsulator layer 19 electrically insulates the cell liquid located in theinner cavity 16 from the electroporation electrode 7.

It should also be mentioned that the measuring electrode 6 and/or thewall that delimits the inner cavity 16 of the measuring andelectroporation electrode(s) 6,7 can have a surface roughness whichenlarges the surface of the electrode. The electrode(s) 6,7 can, forexample, consist of porous silicon or a coating made of this material.

As can be recognized especially well from FIGS. 1 to 10, the electricinsulator 9 has within the support area 5 a projection 20 that projectsbeyond its surface plane, on which projection is arranged the free endof the active electrode area of the electroporation electrode 7, whichfaces away from the surface plane. The active electrode area 8 is thuselectrically well coupled to the cell 3 that adheres to the supportarea. The cross section of the projection 20 tapers going away from thesurface plane of the support area 5 to the point that projects thefurthest outwardly. The projection 20 and the electrode(s) 6,7 can thusbe manufactured with better production engineering. Moreover, thetapering projection 20 has a good mechanical rigidity.

The projection 20 can, however, also have a cross section that isconstant in its extension direction or decreases going away from thesurface plane of the support area 5 to the furthest projecting point.Such a projection 20 can, for example, be manufactured usingLIGA-technology.

It should also be mentioned that the specimen slide 4 has asubstantially plate-shaped substrate 21 which, for example, can consistof a semiconductor material (e.g. silicon or gallium-arsenide), siliconcarbide, glass or plastic. On this substrate the insulator 9 can beapplied as a coating, for example by sputtering. Optionally, thesubstrate 21 can also be a flexible film.

In the embodiment according to FIGS. 11 to 18, the specimen slide 4 hasin the support area 5 respectively several profile recesses 22surrounding the electroporation electrode 7. As can be recognizedespecially well from FIG. 11, the seal of the cell membrane of the cell3 against the support area 5 of the specimen slide 4 can be improved bythis.

In the embodiment according to FIGS. 11 to 14, the profile recesses areclosed ring grooves, which are arranged concentrically to theelectroporation electrode 7. The ring grooves each have an approximatelyrectangular shaped cross section. Ring grooves adjacent to each otherare arranged respectively at equal distances from each other (FIG. 12).The distances between adjacent profile recesses 22 and the depth ofthese profile recesses 22 are adapted to the type of cells 3 to besupported on the support area 5. The edges of the profile recesses 22can be rounded in order to make the adherent supporting of a cell 3easier.

The profile recesses 22 can have gaps in their course, as is shown inthe example of a checkerboard structure in FIG. 15 and a honeycombstructure in FIG. 16. The surface profiles according to FIGS. 14 to 16,the surface roughness, and the surface material can be respectivelyadapted to a certain cellular type. In this way, the cell adhesion canbe improved or controlled.

In the embodiments according to FIGS. 11 and 18, the surface profile canbe applied on the electric insulator 9 as a coating using methods ofsemiconductor technology. The specimen slide 4 can thus be manufacturedin a simple way as a semiconductor chip. The surface profile can,however, also be applied on the insulator 9 with other processes, forexample in thick layer technology. In the embodiment according to FIG.13, the surface profiling is a layer applied on the substrate 21, whichis covered by an insulator layer which forms the insulator 9. By thismeasure, possible creep (leaking) currents, which flow from theelectroporation electrode 7 along the surface of the insulator 9 to thenutrient medium 2, can be reduced. A suitably high electric resistancethereby results between the electrode 6,7 and the nutrient medium 2,when a cell 3 is supported on the support area 5.

The manufacture of the surface profile shown in FIG. 13 can beaccomplished, for example, in a manner where on the substrate 21 a ringstructure is applied using a mask technique, which is then coated withthe insulation layer. The profiling then has, in comparison to theembodiment according to FIG. 11, somewhat more rounded edges. The cells3 can then be supported better.

In the embodiment according to FIG. 18, boundary walls 40 are arrangedon both sides of the electrode, which walls form together with theinsulator 9 a guide channel 41 that is somewhat U-shaped incross-section. In this, the profiles 22 and the measuring electrode onthe floor of the guide channel 41 are arranged between the boundarywalls 40. The boundary walls 40 form an obstruction for cells 3 locatedin the guide channel 41, which these cells cannot or not easilysurmount. The cells 3 can thus essentially only move in the extensiondirection of the guide channel 41, where they forcibly come into contactwith the measuring electrode 6. The clear spacing of the boundary walls40 arranged on both sides of the measuring electrode 6 is adapted to thedimensions of the cells 3 and is preferably somewhat larger than thediameter of the cells 3. Optionally, several measuring electrodes 6 canbe arranged one behind the other in the extension direction of the guidechannel 41. In this way, the cell potential can be measured on severalcells 3 at the same time. The cross-section of the guide channel 41 cantaper or expand in the extension direction, i.e. the guide channel 41can have a different width and/or different cross-sectional dimensionsat different positions. Starting from the deepest to thewidest-projecting point of the guide channel 41, the cross section ofthe guide channel 41 can taper, for example.

In the embodiment according to FIG. 9, a junction-field effecttransistor (J-FET) 23 is integrated into the substrate 21 of thespecimen slide 4. Directly above the gate of the J-FET, the measuringand electroporation electrode 6,7 is arranged, which is connected withthe gate in a galvanically conducting manner. An additionalgate-electrode 30 allows an external control of the channelconductivity, which makes possible an operating point adjustment Source24 and drain 25 of the J-FET 23 are connected via conductor pathsintegrated into the specimen slide 4 with an evaluation device. TheJ-FET 23 has a very low noise, a high input impedance, as well as a lowinput capacitance, and causes an impedance transformation of theelectric signal that is uncoupled from the cell 3. With the J-FET 23,the measuring and electroporation electrode 6,7 is for the most partuncoupled from the conducting capacitance of the conductor pathsconnected to the source 24 and the gate 25. In this way, high-frequencysignal portions of the cell potential signal can be better measured.

For the electroporation of the cell membrane, the electroporationelectrode 7 can be connected with a conductor path 17 integrated in thespecimen slide 4 to an electroporation voltage source 43. Instead ofcoupling the electroporation voltage via the conductor path 17, theelectroporation voltage can also be coupled capacitively via the gate ofthe J-FET 23 into the electrode 6,7, in which case the conductor paths44 connected to the source 24 and the drain 25, and possibly thesubstrate 21, are connected to the electroporation voltage source. Inthis case, the conductor path 17 can be omitted.

In the embodiment according to FIG. 10, a switch-FET 26 is integratedinto the specimen slide 4 in close proximity to the J-FET 23. The drainconnection 27 of this switch-FET 26 is connected to the electroporationvoltage source 43 and the source connection 28 to the electrode 6,7. Toapply the electroporation voltage to the electrode 6,7, the gate of theswitch-FET 26 is connected to a control line 29, onto which a controlvoltage is applied. In an advantageous way, the electrode 6,7 is to thegreatest extent uncoupled from leaking capacitance and couplings of theconnection line to the electroporation voltage source by the blockedsource-drain connection.

In the embodiment according to FIG. 17, in the support area 5 of thespecimen slide 4, several measuring and electroporation electrodes 6,7are arranged in the form of an array. The individual electrodes 6,7 arerespectively arranged at grid points of a Cartesian coordinate system.In this way, a locationally triggered measurement of the electric signalof a cell population supported on the support area 5 is possible. Theelectrodes 6,7 can also be distributed in other ways in the support area5, for example in rows or columns set apart from each other or randomlydistributed.

On and between the electrodes 6,7, in order to optimize the growth rateof the cells 3, conductive structures can be arranged, which also makespossible a targeted supporting of cells 3 and/or between the electrodes6,7. The conducting structures can, for example, contain a surfacestructuring, a coating or a suitable topography formation. For differentcell types or measuring tasks, different distances between adjacentactive electrode areas 8 can be provided.

On the whole, a method thus results for measuring the cell potential ofa biological cell 3, in which the cell in a nutrient medium 2 issupported adhering to a support area 5. Within the support area 5 of thecell 3, at a distance from the support edge, an opening is made in themembrane of the cell 3. In the process, the edge of the cell membrane,that adheres to the support area 5 and borders the opening, seals thecell liquid located inside the cell 3 against the nutrient medium 2.Through the opening, the electric voltage is measured between the cellliquid and the nutrient medium 2.

In the embodiment according to FIG. 19, a piezo element 31 is arrangedbetween the measuring electrode 6 and the specimen slide 4. The piezoelement carries the measuring electrode 6 on its free end, that can bemoved relative to the specimen slide 4, and is affixed on the substrate21 of the specimen slide 4 by its end facing away from the free end. Asin the embodiment according to FIGS. 5 and 6, the measuring electrode 6is constructed approximately in a truncated cone shape and has anessentially cylindrical inner cavity 16 that extends along the axis ofthe truncated cone. This inner cavity 16 has on the free end of themeasuring electrode facing away from the piezo element 31 a circularopening which is bordered by a ring-shaped electrode area, which has asharp edge 32 facing away from the support area 5. Using the piezoelement 31, the edge 32 of the measuring electrode 6 can be movedrelative to the specimen slide 4 toward and away from a cell 3 that issupported on the support area 5. In this way, an approximately circulardisc-shaped area of the cell membrane is cut out of the membranestructure of the cell 3, so that cell liquid located inside of the cell3 can pass through the resulting opening to come into contact with themeasuring electrode 6. The inner cavity 16 of the measuring electrode 6is filled with an electrolyte by which ions contained in the cell liquidcan proceed to the inner walls of the measuring electrode 6 borderingthe inner cavity 16, after the opening is made in the cell membrane. Inthis way, a good electrical contact between the cell liquid and themeasuring electrode 6 is made.

In the support area 5 an electric insulator surrounding the measuringelectrode 6 is arranged, which insulator is brought via the coneenvelope surface of the measuring electrode 6 right up to the sharp edge32. The cell 3 supported on the support area 5 adheres by its cellmembrane to the insulator 9, whereby the edge of the cell membranesurrounding the opening made in the cell membrane seals off the cellliquid located inside the cell 3 from the nutrient medium 2 in anelectrically insulating manner. To measure the cell potential, themeasuring electrode 6 is connected via a conductor path 18 integrated inthe specimen slide 4 to a measurement amplifier 12. A referenceelectrode 15 (as shown in FIG. 11, but not shown in FIG. 19) located incontact with the nutrient medium 2 functions for determining a referencepotential. The conductor path 18 and the insulator 9 consist of anelastic material which allows a relative movement between the measuringelectrode 6 and the substrate 21 of the specimen slide 4 upon atriggering of the piezo element via the control lines 42.

In the embodiment according to FIG. 20, a laser diode 33 is provided formaking a hole in the cell membrane of a cell 3 supported on the supportarea 5. The laser diode is arranged, as observed from the support area5, behind an optical window 34 provided in the measuring electrode 6 andintegrated into the substrate 21. The laser diode 33 is connected usingconnection lines 37 to a power supply and control device (not shown).The optical window 34 is constructed as a through-passage hole, whichpenetrates the measuring electrode 6 approximately in a direction normalto the surface of the support area 5. Using the laser diode 33, aportion of the cell membrane of the biological cell 3, supported on thesupport area 5 and the measuring electrode 6, can be irradiated with alaser beam, which absorbs the cell membrane of the cell 3. In this way,an opening is made in the cell membrane. After making the opening, thelaser beam is turned off, so that then the cell potential can bemeasured through the opening using the measuring electrode 6. As in theembodiment according to FIG. 19, the cell 3 adhering to the insulator 9bordering the measuring electrode 6 seals the measuring electrode 6 offagainst the nutrient medium 2.

In the embodiment according to FIG. 21, an external laser 35 is providedfor making the opening in the cell 3 that adheres to the support area 5.The laser 35 has a beam that can be coupled to the rear side of thespecimen slide 4 facing away from the measuring electrode 6, for exampleusing a beam guide device 36 which can include an optical light guideand/or a deflection mirror as well as possibly a focusing device. Thebeam passes through an optical window provided in the substrate 21 andinto the hole in the cell 3 that is penetrated by the measuringelectrode 6.

In the embodiment according to FIG. 22, the inner cavity 16 of themeasuring electrode 6 is connected to a liquid channel 38, which can beimpinged by a controllable partial vacuum. The liquid channel 38 can,for this purpose, be connected to a micropump, for example, by which thenutrient medium 2 can be sucked out from the support area 5 of thespecimen slide 4. In this way, the mounting of a cell 3 on the measuringelectrode 6 is made easier. Optionally, after the supporting on themeasuring electrode 6 of a cell that is suctioned via the liquid channel38, a weak partial vacuum can be exerted on the cell 3 for a certaintime period, until it automatically adheres to the support area 5.

As in the embodiment according to FIG. 19, the edge of the measuringelectrode 6 surrounding the opening of the inner cavity 16 has aring-shaped sharp edge 32, which projects beyond the surface plane ofthe support area 5. After supporting the cell 3 on the measuringelectrode 6, a portion of the cell membrane is impinged by a partialvacuum for a short time, by suctioning off nutrient medium 2 at theliquid channel 38, to such an extent that the membrane area bordered bythe sharp edge 32 of the measuring electrode 6 is detached from themembrane structure. In this way, an opening is made in the cellmembrane, through which the measuring electrode 6 can come into contactwith the cell liquid located inside the cell 3. After making theopening, the partial vacuum is switched off in the liquid channel 38.Optionally, after making the opening, a small quantity of cell liquidcan be suctioned into the liquid channel 38 so that it comes intocontact with microsensors 39 arranged in the liquid channel 38 adjacentto the measuring electrode 6. In this way, additional cell parameters,for example the gas content and/or an ion concentration of the cellliquid, can be measured.

After making the opening in the membrane of the cell 3, the transportdirection of the micropump connected to the liquid channel 38 can bereversed for a short time, in order to inject a substance found in theliquid channel 38, for example a medication and/or a fluorescentcoloring agent, through the opening of the cell membrane directly intothe cell interior. If no cell 3 is supported on the electrode 6, theliquid channel 38 can nevertheless be used in order to add anappropriate substance to the nutrient medium 2.

In the embodiment according to FIG. 18, in an approximately ringshapedarea around the free end of the measuring electrode 6 constructed as asharp tip and projecting beyond the surface plane of the support area 5,a chemical substance is immobilized which, upon contact with a cell 3supported in the support area 5, makes an opening in the membrane of thecell 3 . Through this opening, the tip of the measuring electrode 6 cancome into contact with the liquid of the cell 3. The apparatus 1 has aparticularly simple construction.

It will be appreciated by those skilled in the art that changes could bemade to the embodiment(s) described above without departing from thebroad inventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiment(s) disclosed, butit is intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

We claim:
 1. An apparatus for measuring a state variable of at least onebiological cell (3) located in a nutrient medium (2), comprising aspecimen slide (4) having a support area (5) or which the cell (3) issupportable in an adherent manner, at least one measuring probe forcontact with a liquid located inside the cell (3) for measuring thestate variable, and a measurement amplifier 12 allocated to themeasuring probe, wherein the measuring probe is arranged within thesupport area (5) with an electric insulator (9) surrounding it, suchthat the cell (3) is supportable on the insulator (9) to seal it fromthe nutrient medium (2), and at least one poration tool is arranged inthe support area (5) tor forming an opening in a membrane of the cell(3), said opening being in an area of the measuring probe.
 2. Theapparatus according to claim 1, wherein the poration tool is arrangedsubstantially concentrically around the measuring probe.
 3. Theapparatus according to claim 1, wherein the measuring probe comprises ameasuring electrode (6), to which is allocated at least one referenceelectrode (15) contactable with the nutrient medium (2) for measuring apotential of the cell (3).
 4. The apparatus according to claim 3,wherein the measuring electrode (6) includes an electroporationelectrode (7) connectable to an electric voltage source forelectroporation of the cell membrane, and the electroporation electrode(6,7) has at least one active electrode area (8) arranged within thesupport area (5) and surrounded by the electric insulator (9).
 5. Theapparatus according to claim 1, wherein the poration tool comprises anelectroporation electrode (7) spaced from the measuring probe, theelectroporation electrode being connectable to an electric voltagesource for electroporation of the cell membrane, the electroporationelectrode (7) having at least one active electrode area (8) arrangedwithin the support area (5) and surrounded by the electric insulator(9).
 6. The apparatus according to claim 4, wherein the active electrodearea (8) of the electroporation electrode (6,7) has at least one sharptip and is arranged to project beyond a surface plane of the supportarea (5).
 7. The apparatus according to claim 4, wherein theelectroporation electrode (7) comprises a hollow electrode having aninner cavity (16) with an opening located on a surface of the supportarea (5), and the measuring electrode (6) comprises a rod electrodearranged in the inner cavity (16) of the electroporation electrode (7),a free end of the rod electrode extending up to the opening of the innercavity (16).
 8. The apparatus according to claim 4, further comprising astatic switching element arranged immediately adjacent to theelectroporation-electrode (7) for connecting the electroporationelectrode (7) to an electroporation voltage source.
 9. The apparatusaccording to claim 4, further comprising at least one actuator formoving the poration tool across the surface of the support area (5)relative to the specimen slide (4), the actuator comprising a piezoelement (31).
 10. The apparatus according to claim 9, wherein theporation tool has at least one sharp tip (32) contactable with the cellmembrane.
 11. The apparatus according to claim 9, wherein the actuatoris connected to a control device for generating an ultrasonic vibration.12. The apparatus according to claim 9, wherein the measuring probe issimultaneously also the poration tool and is movable across the surfaceof the support area (5) relative to the specimen slide (4) using thepiezo element (31).
 13. The apparatus according to claim 1, wherein thespecimen slide (4) has an optical window (34) in an area of themeasuring probe, the window being arranged in a beam path of a laserbeam in order to open the cell membrane.
 14. The apparatus according toclaim 13, further comprising a laser diode integrated into the specimenslide (4) to generate the laser beam.
 15. The apparatus according toclaim 13, wherein the measuring probe is arranged substantiallyconcentrically around the optical window (34).
 16. The apparatusaccording to claim 1, wherein the poration tool comprises one of achemical substance and at least one outlet opening connected to a supplychannel for a chemical substance for opening the cell membrane.
 17. Theapparatus according to claim 1, wherein the poration tool has at leastone channel opening into the support area (5) for admitting a partialvacuum or excess pressure to impinge upon a portion of the cell membraneto form the opening in the cell membrane.
 18. The apparatus according toclaim 1, wherein the measuring probe comprises a hollow sensor (16)installed in a surface of the specimen slide (4) and having at least oneinner cavity (16), wherein the inner cavity (16) has an opening on thesurface of the support area (5).
 19. The apparatus according to claim 1,wherein the electric insulator (9) within the support area (5) has aprojection (20) that projects beyond a surface plane of the support area(5), and the measuring probe is arranged on a free end of the projection(20) facing away from the surface of the support area (5).
 20. Theapparatus according to claim 19, wherein a cross-section of theprojection (20) tapers from the surface plane of the support area (5) toa point furthest from the plane.
 21. The apparatus according to claim 1,wherein the specimen slide (4) has a profile in the support area (5),the profile having at least one profile recess (22) and/or projectionsurrounding the measuring probe.
 22. The apparatus according to claim21, wherein the profile recess (22) and/or projection is interrupted inits extension direction by at least one gap.
 23. The apparatus accordingto claim 21, wherein the profile recess (22) and/or projection has aring-shape, and a plurality of ring-shaped profile recesses (22) and/orprojections are arranged substantially concentrically to the measuringprobe.
 24. The apparatus according to claim 21, wherein the insulator(9) is an insulation layer arranged on a surface of the profile.
 25. Theapparatus according to claim 21, wherein a profile projection(s) is(are) mounted on a surface of the insulator (9).
 26. The apparatusaccording to claim 1, further comprising at least one coating arrangedin the support area (5) of the specimen slide (4) and applied on itssurface, the coating being selected from the group consisting of a celladhesion protein, a hydrophilic coating, and a hydrophobic coatingimmediately adjacent to the measuring probe.
 27. The apparatus accordingto claim 1, further comprising boundary walls arranged on both sides ofthe measuring probe as a mechanical guide for the cells (3), theboundary walls delimiting a groove-shaped guide channel (41).
 28. Theapparatus according to claim 1, further comprising a field effecttransistor (FET) (23) arranged adjacent to the measuring probe (6),wherein the measuring probe is connected to a gate of the FET (23) forimpedance transformation of a measurement signal.
 29. The apparatusaccording to claim 1, further comprising at least one liquid channel(38) opening in the support area (5) of the specimen slide (4), thechannel being surrounded by the measuring probe.
 30. The apparatusaccording to claim 29, wherein at least one micropump is integrated inthe specimen slide (4) in the course of the liquid channel (38).
 31. Theapparatus according to claim 30, further comprising at least onemicrosensor (39) arranged within a wall of the liquid channel (38) formeasuring a quantity of the cell (3).
 32. The apparatus according toclaim 1, further comprising at least one additional electrode arrangedin or adjacent to the support area (5) to generate an electric fieldconnecting the cell (3) to the measuring probe.
 33. The apparatusaccording to claim 1, wherein several measuring probes are arranged asan array in the support area and to each of these measuring probes atleast one poration tool is allocated.